Sequence analysis and plasmid mobilization of a 6.6-kb kanamycin resistance plasmid, pSNC3-Kan, from a Salmonella enterica serotype Newport isolate

Research on the transfer of antibiotic resistance plasmids has been mainly focused on the large multi-drug resistance conjugative plasmids, while the transmission of small mobilizable plasmids remains under-investigated. A series of diverse ColE-like kanamycin resistance plasmids (“KanR plasmids”) from Salmonella enterica were characterized previously. In this study, the 6.6-kb pSNC3-Kan from a Salmonella enterica serotype Newport isolate was investigated. It possessed highly conserved RNA I/II and Tn602 (IS903-aph-IS903) regions to two other KanR plasmids pSe-Kan and pSBardo-Kan, but carried a mobC-mobA/BD operon. The mobilization proteins encoded by the mob operon of pSNC3-Kan showed high sequence identity (~95%) to those of an E. coli plasmid pEC34B, except that MobE was not present; and were much less conserved to those of another KanR plasmid pSN11/00Kan (43% - 86% identity). Four structurally different KanR plasmids were investigated for their ability to be mobilized by the conjugal transfer (tra) genes from F and IncP plasmids. Transfer genes derived from IncP plasmids can efficiently mobilize KanR plasmids possessing the mob operons (mobC-mobA/BD), such as pSNC3-Kan and pSN11/00Kan, in bi-parental mating experiments. On the other hand, F tra genes were able to mobilize pU302S, pSNC3-Kan and pSe-Kan, but not pSN11/00Kan. A plasmid-borne mob operon was not required for mobilization of the oriT(F)-bearing pSe-Kan by the F tra genes. This study underscores the complexity of plasmid interaction and the importance of how small mobilizable plasmids may contribute to the spread of antibiotic resistance genes.


Introduction
Outbreaks involving Salmonella enterica accounted for 30% of foodborne diseases of known etiology that occurred between 2009-2015 in the United States [1]. They are responsible for an estimated 1.2 million annual salmonellosis cases just in the U.S. alone and account for both leading causes of hospitalization and deaths among the major foodborne pathogens [2,3]. these KanR plasmids, we present here the sequence analysis of the pSNC3-Kan plasmid from S. Newport strain ARS #574, and evaluation of the mobilization of four representative KanR plasmids by F and IncP conjugal transfer systems.

KanR plasmid mobilization experiments
Detailed information on the bacterial strains and plasmids used in this study are listed in Table 1. To generate the donor strains for bi-parental mating: KanR plasmids (purified from DH5α host) were transformed into NEB5αF'I q (carrying F'; NEB; library-efficiency competent cells) or S17-1λpir (RP4/IncP tra gene integrated into the chromosome; gift from Dr. Clay Fuqua, Indiana University, Bloomington, IN; [29]). Mating procedure was modified from that used by Brasch and Meyer [30]. Briefly, cells were grown overnight at 37˚C in LB with required antibiotic. On the day of mating, cells were sub-cultured in LB without antibiotics and grown to mid-log phase (OD 600~0 .3-0.4); cells were then washed, resuspended in LB, and spotted on a LB agar plate at 1:10 (donor: recipient) ratio. Mixtures were allowed to dry at room temperature briefly (~15 min), and then incubated at 37˚C for 60 min. Cells were collected from the mating plate, resuspended in LB, and serially diluted and plated on LB agar supplemented with antibiotics using 6×6 drop plate methods [31]. Donor strain S17-1λpir carrying KanR plasmid was paired with recipient CAG18483 (Tet R ) and transconjugants were selected on LB supplemented with Kan 50 μg/mL and Tet 2 μg/mL.

Sequence features of pSNC3-Kan
The pSNC3-Kan plasmid was determined to be 6,606 bps, 51.6% G+C. Sequence was deposited in NCBI GenBank under accession # MW030687. Plasmid features are listed in Table 2. It is comprised of RNA I/II region, rom, mobC-mobA/BD operon, and carried the "IS903-aph (3')-I-IS903 cassette" (Tn602; the two tnp genes arranged in the same orientation; [32,33]), which is an indicative feature of the group C/C2 plasmids previously reported [20]. pSNC3-Kan was previously partially sequenced and assumed to be a variant of the group C plasmids due to their identical RNA I/II region and the aph(3')-I gene, in addition to their similar restriction digest patterns. Yet to our surprise we found an operon of mobilization genes  Tn602. The Tn602 of pSNC3-Kan was 2-bp and 1-bp different from those of the pSe-Kan and pSBardo-Kan plasmids, respectively. The two IS903 (isoform IS602) copies on pSNC3-Kan were 22-bp different from each other. The occurrence of Tn602 is relatively rare and has been found on just 3 different plasmid backbones in nature: on IncFII plasmid pDG10, B/O plasmid R805a, and these KanR plasmids [32]. Upon re-examination of the pSe-Kan sequence, we identified the 9-bp target duplications "GTTGCTAAT" flanking the Tn602 at coordinates 1-9 and 4060-4068; same target duplications were also found flanking the Tn602 on pSBardo-Kan (nt 1-9 and 5126-5134). This was also pointed out by Moran and colleagues on their analysis of the Tn602 on a large B/O plasmid R805a in S. Typhi [32]. However, there was only one such sequence in pSNC3-Kan at nt 1-9. It is conceivable that the Tn602 was acquired by a common ancestor of the group C/C2/C3 plasmids possessing a mob operon, and then a recombination event resulted in the loss of the mob genes and replaced by the 3 ORFs of unknown function with low %G+C (~35%), resulting in a pSe-Kan-like plasmid. A BLASTN search (conducted on September 9, 2019) found one small plasmid in E. coli O111:H-strain 110512 (pO111-110512_6; AP019767) carrying the same 3 unknown ORFs with 99% identity to those of pSe-Kan and pSBardo-Kan; this was the only plasmid other than the group C/C2 KanR plasmids shown to possess such sequences.
RNA I/II and Rom. The RNA I/II region of pSNC3-Kan (nt 127-670) is 100% identical to those of group C/C2 plasmids pSe-Kan and pSBardo-Kan, and shared 98.6% and 97% identity to pU302S (group A) and pSN11/00Kan (group B), respectively. PlasmidFinder (v.2.1) did not identify any Col replicon using the default setting (95% identity/ 60% coverage). It was classified as a ColRNAI replicon only when the threshold settings were relaxed (89.6% to pIGMS32, coverage 125/130). Somewhat surprisingly, we found that the RNA I modulator protein (Rom) of pSNC3-Kan is more similar to that of the group B plasmid pSN11/00Kan (95.2% identity), than to the other group C/C2 plasmid pSe-Kan (61.9% identity). Alignment of Rom proteins are shown in Fig 2A. These findings agreed with the pairwise alignment that the sequence homology between pSNC3-Kan and C/C2 plasmids ends at nt 1038, about half-way between the first oriT and rom (as shown in Fig 1A).
Megablast against nr database found other plasmids with extremely high identity in the RNA I/II region, including E. coli plasmids pEC16II (99.1% identity/86.4% query coverage; KU932034; 7939-bp), and pEC34B (98.9% identity/100% query coverage; HQ622576; 6982-bp). Plasmid pEC34B from E. coli serotype O34 strain NCTC 9034 was conserved over the entire query length and well-annotated, thus was chosen for further comparison. Pairwise alignment of pSNC3-Kan against pEC34B showed homology spanning~50% of the plasmid (>3.1-kb), from the BamHI site (nt 83-88) extending to the end of the mobA, with 95.5% overall nucleotide identity; however, pSNC3-Kan did not possess a mobE gene downstream of mobA, as was the case in pEC34B (Fig 1A).
Mobilization proteins of pSNC3-Kan. Mobilization protein sequences from pSNC3-Kan, pSN11/00Kan, and pEC34B were aligned in Geneious (default setting: Global alignment with free end gap, cost matrix: Blosum62). The alignments and pairwise identity of the mobilization proteins MobC, MobA, MobB and MobD are shown in Fig 2B. Accessory DNA binding proteins containing the ribbon-helix-helix (RHH) domain(s) are involved in specific sequence recognition and binding near the nic site at oriT to help relaxase to form relaxosome and stimulate nickase function (for review, see [9,15]). MobC, the mobilization accessory protein, of pSNC3-Kan was better conserved (86.09% identity) to that of pSN11/ 00Kan than the MobA relaxase and the internally encoded MobB and MobD (identity of 60.71%, 43.01%, and 68.83%, respectively); much greater protein homology (~95%) was observed to those of pEC34B. The N-terminal third (~155 residues) of the MobA proteins are highly conserved between those of pSN11/00Kan and pEC34B; the conserved Y19, SF and HQD-x4-ELNF relaxase motifs and residues [34] are all present in MobA_pSNC3-Kan. The low protein homology observed in the C-terminal two-thirds of the MobA, as well as MobB and MobD, between pSNC3-Kan and pSN11/00Kan strongly suggests that the mob region from these two plasmid groups were derived from different lineages.
Megablast of the mobA gene against nr database showed a considerable number of hits (94 of the 283) with >90% query coverage. The majority of those hits were in E. coli, with the maximum identity of 98% (100% query coverage) to an unannotated 7,991-bp E. coli plasmid p720632_7 (CP025843). The BLASTP top hits included proteins on E. coli plasmids pEC34B (96.6%; HQ622576), pLG3 (AF251289; 95.4%), and p5217 (NC_011799; 96.5%), although the max. identity was 100% (with 100% coverage) to proteins annotated as E. coli DNA polymerase (WP_047662141.1; KHH54811.1, KHH60013.1), or nuclease (MDL80525.1, MFR30254.1). The fact that pSNC3-Kan shares an extensive, conserved region spanning from RNA I/II to mobC-mobA/BD with the E. coli pEC34B plasmid, as well as having higher homologies to E. coli Mob proteins than to those of other S. enterica plasmids, may suggest that these two plasmids shared a recent common ancestor, and that pSNC3-Kan (and the group C ancestral plasmid) were recently acquired by Salmonella from E. coli. It is noteworthy that although the two plasmids are highly similar, the homology between pSNC3-Kan and pEC34B ended after the stop codon of mobA; the mobE gene was not present on pSNC3-Kan. The location of the potential XerC/XerD binding sites of the two plasmids were not conserved.
Origin of transfer (oriT). The oriT region was predicted to be at nt 913-1001 in pSNC3-Kan based on the annotation of pEC34B, although the oriTFinder identified a slightly shorter oriT at nt 912-997, with an H-value of 0.87 to that of pEC886 (oriTDB accession # 100129). The oriTFinder located the oriT on another KanR plasmid pSN11/00Kan at nt 989-1071 with a higher H-value of 0.93 to pEC34B (oriTDB accession # 100130). Sequence alignment and pairwise identity of the closely-related oriT regions of the KanR plasmids (excluding pU302S), plus those of the pEC34B, pEC886 and ColE1, are presented in Fig 3. Importantly, the putative 269-bp oriT(F-type) [labeled as "oriT2_pSe-Kan" in oriTDB, accession # 100060] found in the C/C2 plasmids (nt 1522-1790) was not identified in pSNC3-Kan.
Phylogenetic analysis of KanR plasmids. Multiple sequence alignment of nucleotide sequences of the five KanR plasmids, pKPN2 [35], ColE1 [36], pEC34B and two other related natural plasmids carrying aph(3')-I (NTP16 [37] and pUB2380) was analyzed using MUSCLE and the resulting Neighbor-joining cladogram is shown in Fig 4. Information on the plasmids used in the alignment is listed in S1 Table. Group C/C2/C3 KanR plasmids were clustered on a separate branch from the group A and B plasmids pU302S and pSN11/00Kan, respectively. pKPN2, which showed an extended homologous region with the backbone of group C/C2/C3 KanR plasmids, also grouped appropriately. NTP16, possessing the nikA and aph(3')-I gene, clustered with the nikA-bearing pU302S. pUB2380, a plasmid carrying aph(3')-I and the mob region previously found in the BLASTN search of pSN11/00Kan [19], is on the same branch with pSN11/00Kan. pEC34B and ColE1 fell on a separate branch from the rest of the plasmids in this alignment, likely due to the differences in the cargo genes.
Plasmids are subject to continuous evolution/rearrangements: recombination, insertion/ deletion, or acquiring genes from other mobile elements and chromosomes via transposons and insertion elements. This analysis is mainly to show the relatedness of the KanR plasmids, and does not necessarily imply the actual ancestry of these plasmids. The KanR plasmid Consensus is shown as the majority of the aligned sequences. "." denotes identical amino acid sequence to that of the consensus. "-" indicates gap in alignment. "X" denotes not conserved residue (in consensus). "?" denotes the position with gap and not conserved residue (in consensus).
https://doi.org/10.1371/journal.pone.0268502.g002 Sequence labels started with "oriT" were retrieved from the oriTFinder database oriTDB and accession numbers are indicated within the square brackets in the pairwise comparison table. pSe-Kan oriT region was annotated based on the alignment to pEC34B, and is extended beyond the "oriT1_pSe-Kan" in oriTDB (shown as light blue box under the sequence). ColE1 sequence was extended to the same span as that of "oriT_pEC34B"; the "oriT_ColE1" in oriTDB is shown as light blue box under the sequence. https://doi.org/10.1371/journal.pone.0268502.g003

Fig 4. Cladogram of KanR plasmids and other plasmids.
Neighbor-joining tree (without distance corrections) generated from multiple sequence alignment using MUSCLE. Plasmid name is followed by GenBank accession number (nucleotide sequence); branch length is indicated within the parentheses. Relevant information on the plasmids used in the alignment is listed in the S1 Table. https://doi.org/10.1371/journal.pone.0268502.g004

PLOS ONE
grouping based on the restriction patterns corresponded well with the sequence comparison and phylogenetic relationship. The extremely high sequence identity of the three group C/C2/ C3 plasmids strongly suggests that the divergence of these plasmids was a relatively recent event. On the other hand, these plasmids were distributed in several S. enterica serovars including Typhimurium (pSe-Kan), Bardo (pSBardo-Kan), Newport (pSNC3-Kan and pSe-Kan), Orion (pSe-Kan) and Brandenburg (pSNC3-Kan), isolated from cattle, dairy cattle or turkey [16,17]. Although pSBardo-Kan was only isolated once, pSe-Kan and pSNC3-Kan were isolated multiple times from more than one serovar and different animal source, implicating the spread of these plasmids within S. enterica.

Mobilization of KanR plasmids by tra genes of F and IncP plasmids
Mobilization of four representative KanR plasmids by the conjugal transfer (tra) genes derived from the F and IncP plasmid was evaluated using bi-parental mating. Many of the available IncP plasmids carried kanamycin resistance genes and thus could not be utilized. After an extensive search we opted to use the readily available, well-established S17-1λpir strain to supply the required IncP tra genes. S17-1λpir has the RP4-2 (Km::Tn7, Tc::Mu-1) plasmid integrated into the chromosome and is susceptible to Kan, Tet and Amp; the strain is commonly used to mobilize shuttle vectors via bi-parental mating [29]. Conjugal transfer genes of the F plasmid were supplied by the F' plasmid in NEB5αF'I q . Results of the plasmid mobilization experiments are presented in Table 3. The S17-1λpir donor-only controls showed~2-fold increase after the 60-min incubation period, while NEB5αF'I q donor-only control remained about the same (0.8-to 1.2-fold change). When no plasmid transfer occurred (indicated as "< 1x10 -7 " in Table 3), the total Kan-resistant counts were the same as the donor-only control counts. We evaluated the mobilization results based on the number of transconjugants in total kanamycin-resistant cells to simplify the experimental planning/logistics. This approach makes sense from the population perspective since the goal is to assess the contribution of plasmid transfer influencing the total antibiotic resistant population.
Mobilization by IncP tra genes. KanR plasmids possessing the ColE-type oriT and mob operons (pSN11/00Kan and pSNC3-Kan) were mobilized readily by the IncP tra genes, with over two-thirds of the Kan-resistant cells carrying mobilized KanR plasmid (Table 3). IncP tra genes could not mobilize pSe-Kan or pU302S, both without a mob operon. Although plasmids pSe-Kan and pSNC3-Kan share similar oriT (ColE-like) regions, pSe-Kan lacks the mob operon and no other mob genes were present in the donor cells, thus confirming that mobilization of these ColE-like plasmids by IncP tra genes require the mobilization accessory genes and a cis-acting oriT. Mobilization by F tra genes. F tra genes mobilized plasmids pSNC3-Kan and pSe-Kan efficiently, but mobilization of pU302S occurred very infrequently (Table 3). High variability between experiments was observed for all 3 mobilizable KanR plasmids by F', particularly for the pSNC3-Kan dataset which varied by as much as 55-fold (individual results were listed in the footnote of Table 3). F plasmid transfer was shown to be highly regulated and very sensitive to cell growth stage and environmental conditions-the transfer efficiency quickly declined after mid-exponential phase [38]. Although extra care was exercised to keep the OD600 of cultures between 0.3 and 0.5 when culturing the donor cells, we were not able to get maximal mobilization efficiency consistently.
ColE1 derivatives can be mobilized by various conjugative plasmids, especially by IncFI (F'lac pro) very efficiently; this requires the presence of the cis-acting oriT (bom) region and mobilization proteins on the same or other complementing plasmids [10]. The fact that pSe-Kan lacked a mob operon but was still mobilizable by F tra genes, suggests that the oriT(F) may be sufficient to facilitate the binding of the F conjugal transfer proteins without the assistance of other mobilization relaxase and accessory proteins-a scenario of "relaxase-in-trans" [39]. The oriT(F) of pSe-Kan is~90% identical to the F oriT (U01159.2; [40]). The alignment and the binding sites of TraM, TraY, and the Integration host factor (IHF) are shown in Fig 5. The F TraY accessory protein causes DNA bending when bound to sby sites and stimulates the relaxase activity of TraI at the nic site; IHF-mediated DNA bending also alters the ability of relaxases to melt and cleave DNA. While TraM is not required for cleavage, it is important for conjugation, likely through interaction with the TraD coupling protein to move the relaxosome to the conjugative pore [9,15,41]. The two high affinity TraM binding sites sbmA and sbmB assist and stabilize TraM binding to the lowest affinity site sbmC, which is critical for conjugation [41]. Hydroxyl radical footprinting [41] showed two TraM dimer footprints centered at the ACAACA sequence between the inverted repeats at sbmC, where the least conserved region between pSe-Kan and F is located. Although there was no sequence conservation at the sbmC contact sites [41], the significance of these mismatches cannot be ascertained without further research. The oriT(F) of pSe-Kan is 100% identical to another small plasmid pKPN2, which also lacks mob genes (AF300473; [36]). pKPN2 shared high sequence identity (95%) with pSe-Kan in the~2-kb region (nt 81-2039) spanning from RNA I/II to cer, which includes the identical oriT(F) region, and 1-bp mismatch in oriT1(ColE-type) (alignment shown in S1 Fig). pKPN2 was shown to be mobilized by F'(lac-gal) plasmid with high efficiency [36]. When we examined the plasmid minipreps from the transconjugants, the mobilized KanR plasmids were observed in all the plasmid preps. Taken together the highly conserved sequence and similar mobilization results of pKPN2, we think that this further supports our finding that the pSe-Kan plasmid mobilization by F tra genes does not require the plasmid-borne mobilization proteins. However, the possibility that pSe-Kan might form cointegrates with the F' plasmid cannot be completely ruled out.
On the other hand, the failure to mobilize pSN11/00Kan by F tra genes was somewhat puzzling, given that the plasmid possesses a highly conserved oriT to ColE1 (Fig 3) and moderately conserved mobC-mobA/BD operon to those of pSNC3-Kan (Fig 2). The nic site within oriT, the relaxase, and accessory proteins all contribute to the transfer specificity [9]. It is possible that the interaction between the pSN11/00Kan mobilization proteins, oriT, and the F conjugal transfer proteins was not optimal, and may favor other conjugative plasmids such as IncP (see above) or IncI. Further in-depth studies are needed to reveal the precise molecular mechanism (s) involved in the observed differences in mobilization of different KanR plasmids by the tra genes of different conjugative plasmids.
Mobilization of pU302S (group A) by F' was only detected at a very low occurrence. pU302S was present in all the plasmid preps from the transconjugants. It is unlikely that cointegrates were formed between pU302S and F', since a higher transfer efficiency would have been expected. The 108-bp oriT sequence on pU302S is that of the pEC34A-type (oriTDB accession #100131; H-value = 0.97), shared~78% identity to the oriT of IncI1 plasmid R64 but with longer (20-bp) inverted repeats [14]. There was no significant similarity found to the F oriT using BLASTN (Expect value 0.05). Moran and Hall analyzed pCERC7 and related nikAbearing small plasmids, including pU302S and NTP16, and showed that all carried identical NikA (53% identity to that of R64), and extremely conserved oriT (up to 3 mismatches); none of these plasmids carried the NikB relaxase homolog [14]. NikA protein is expected to bind to the inverted repeats in the oriT and interact with the relaxase of an I-complex plasmid to initiate transfer [14]. NTP16 was the only plasmid in this nikA-bearing family that has been shown to be mobilized by R64 [14,37], suggesting that its NikA can interact with the relaxase of R64. The homology of NikA and oriT of this nikA-bearing plasmid family to the IncI1 system is moderate, it is plausible that another yet-to-be-identified conjugative plasmid group may interact with this plasmid family more efficiently. Alternatively, the system may have evolved to be more flexible to work with multiple conjugative systems. Mobilization of this nikA-bearing family by F has never been reported. Although the structure of the pU302S/NTP16 NikA protein has not been determined, it is reasonable to assume that it retains the highly conserved RHH domain structure of NikA_R64 and MbeC_ColE1, as shown by Varsaki and colleagues [34]. Since MbeC_ColE1 can interact with many different conjugative T4SS including those of F and I1, based on our experimental findings we speculate that NikA_pU302S may interact with the conjugative system of F, albeit less efficiently, to initiate transfer. To our knowledge this is the first report of a plasmid bearing nikA to be mobilized by F tra genes. Since F plasmid transfer is sensitive to growth stage, the log phase cultures utilized in our mating experiments may have played a role in optimizing these inefficient mobilization events.

Discussion
Although the original S. Newport isolate ARS# 574 was shown to carry additional IncA/C and I1 replicons and no co-resident IncF or P plasmids [16], other KanR plasmids came from S. enterica isolates carrying IncFI/FII, P, I1, or other replicons [16,[18][19][20]42]. We recently isolated and sequenced several conjugative plasmids belonging to the IncI1, X1 and X4 groups that were able to mobilize at least some of the KanR plasmids using a qualitative tri-parental mating assay [43]. Although not reported in that study, pSNC3-Kan was mobilizable by all the conjugative IncI1, X1 and X4 plasmids identified, in a pattern similar to that of pSN11/00Kan; on the other hand, pU302S and pSe-Kan were mobilizable only by IncI1 plasmids, but not by IncX1 or X4 plasmids [43]. No IncA/C plasmid was identified in the same study [43]. Other attempts to acquire or isolate IncA/C plasmids capable of mobilizing KanR plasmids were also unsuccessful because all available IncA/C plasmids or strains were also resistant to kanamycin.
Ares-Arroyo and colleagues have shown that 74% of the Enterobacteriaceae isolates tested carried at least one ColE-like plasmid using a PCR-based system for detection and capture of plasmid sequences [44]. Cohen and colleagues [45] detected small plasmids (2.2 to 6 kb) in 17 out of 19 MDR S. enterica poultry isolates of a variety of serovars in Israel; some of these also carried a co-resident IncI1 or X1 conjugative plasmid. Among these, a Newport isolate was shown to harbor 3 small plasmids (2.2, 3.2, and 6 kb) and a 44.6-kb IncX1 plasmid; however, mobilization of these small plasmids was not tested [45]. A recent study by Oliva and colleagues [46] showed that two small ColE-like plasmids in a S. Typhimurium MDR strain ST1030 were co-transferred with the large IncI1 conjugative plasmid pST1030-1A at very high frequencies: pST1030-2A (possessed mobC-mobABDE) was found in 92% of the transconjugants and~56% of the transconjugants harbored both small plasmids pST1030-2A and pST1030-3 (which possessed an orphan mob-associated oriT) [39,46]. Another example of "relaxase-in-trans" was reported by Moran and Hall [47]: a 1982-bp cryptic rolling-circle plasmid, pBuzz, which carried two copies of oriT's but without the relaxase or other accessory genes, was mobilized by the co-resident B/O plasmid with high efficiency; other related rolling-circle plasmids may also carry diverse oriTs of different Inc types [47]. These studies, as well as ours, showed that numerous variations exist in the mobilization mechanisms/strategies employed by the small plasmids, many of which do not require a plasmid-borne mobilization relaxase protein. We concur with the notion that the approach using relaxase alone to predict potential plasmid transferability is insufficient, and underestimates the influence of small plasmids in horizontal gene transfer [39,47].

Concluding remarks
Here we demonstrated that ColE-like KanR plasmids can be mobilized with varying efficiency by conjugative plasmids F or IncP in E. coli. Plasmid pSNC3-Kan was the only KanR plasmid that can be mobilized by both F and IncP tra genes. We showed that the F conjugative system is capable of mobilizing pSe-Kan which lacks the mobilizable plasmid-borne relaxase or accessory proteins, likely through the interaction with oriT(F). We also reported the first known case of nikA-bearing plasmid mobilized by F (at low efficiency). However, without mechanistic proof we can only speculate that the NikA and oriT of pU302S may be able to interact with the F tra genes. These naturally occurring kanamycin resistance plasmids with different mobilization accessory proteins and oriT regions are valuable resources in providing insights on how small plasmids are mobilized by different conjugative plasmids. Although studies on the identification and sequencing of small plasmids have increased significantly due to a large number of whole genome sequencing projects, comprehensive annotation of the small plasmids is often lacking. Research on the capability and mechanisms of small plasmid mobilization is still extremely limited and deserves more attention.